Using positional methods, we recently cloned the Lps locus of mice, establishing its identify to the toll-like receptor 4 (Tl4) locus. In all probably, the Tl4 protein represents the transducing subunit of the mammalian receptor for bacterial endotoxin (lipopolysacharide; LPS). Hi homozygous form, destructive mutations of Lps prevent most (and in some cases all) biological responses to LPS. Both the co-dominant Lps allele of C3H/HeJ mice (Lps/d; representing a point mutation of the Tlr4 cytoplasmic domain) and the recessive Lps allele of C57BL/10ScCr mice (representing a deletion encompassing the entire Tlr4 gene) yield phenotypes in which profound unresponsiveness to Lps is coupled with heightened susceptibility to infection by Gram-negative organisms. Our identification os Lps as Tlr4 has opened the door to a wide range of studies in the endotoxin field and beyond. As a limiting factor in LPS signal transduction, it is likely that Tlr4 determines the LPS sensitivity of mononuclear cells. Hence, it may be that well known phenomena such as LPS tolerance, LPS priming and interspecies variability in LPS responses are dependent upon variation in the structure or expression of Tlr4. In this proposal, we outline plans to examine the precise role played by the Tlr4 protein when it participates in LPS sensing. CD14, an upstream component of the LPS signaling pathway, may interact directly with Tlr4; alternatively, there may be other (as yet unknown) proteins interposed. We will search for novel transducing molecules that interact with Tlr4 to carry the LPS signal into the cytoplasm. We will study the regulation of Tlr4, analyzing the effects of agents that strongly modulate sensitivity to LPS. Finally, we will examine the hypothesis that TLR4 is a susceptibility locus affecting the occurrences and/or outcome of severe Gram-negative infections in humans. NOMENCLATURE USED IN THIS PROPOSAL: TLR4, the human toll-like receptor 4 gene; Tlr4, the mouse toll-like receptor 4 gene; Tlr4, the toll-like receptor 4 protein of either species; LPS, lipopolysacharide; LPS, the mouse LPS gene; LPS/d, the LPS allele represented in C3H/HeJ(LPS-unresponsive) mice. Lps/n, the common wild-type Lps allele; Tlr4/Lps-d, the Lps/d allele of Tlr4; Tlr4/Lps-n, the Lps/n allele of Tlr4.